The investigations in this proposal are focused on identifying the molecularbasis for the direct and indirect consequences ofVitamin D Receptor (VDR) ablation. VDRknockoutmice develophypocalcemia and secondary hyperparathyroidism which, in turn, leads to hypophosphatemia. They also develop rickets and osteomalacia. However,when placed on a special diet that maintains normal mineral ion levels, the VDR null mice have histologically, histomorphometrically and biomechanicallynormal long bones, thus the skeletal effects of VDR ablation are indirect and are a consequence of impaired VDRaction in the intestine which results in abnormal mineral ion homeostasis. This observation led us to focus our investigations on the pathophysiological basis for the development of rickets, which is an indirect consequence ofVDR ablation. We demonstrated that the growth plate abnormalities in the VDRnull mice are a consequenceof hypophosphatemia, which leads to expansion ofthe late hypertrophic chondrocyte layer due to impaired apoptosis ofthese cells. Since hypophosphatemia leads to impaired terminal chondrocytedifferentiation during postnatal life, studies are proposed to address the hypothesis that phosphate restriction also impairs chondrocyte differentiation during endochondral skeletal development and skeletal repair. Investigations will be performed using the ex vivo mouse metatarsal culture system and the in vivo femoral fracture repair models. These studies will identify developmental pathways that are interrupted byhypophosphatemia, an indirect consequence ofVDR ablation. Analogous to humans with VDRmutations, VDRnull mice have hair perinatally,but develop alopecia. Hair reconstitution assays and studies in transgenic mice demonstrate that VDRexpression in keratinocytes is both necessary and sufficient for the prevention of alopecia,thus the effects of the VDRin this cell are direct. We have also demonstrated that the actions ofthe VDRrequired to prevent alopecia are ligand- independent, thus reflect novel molecular actions of this nuclear receptor. Investigations in this proposal will address the hypothesis that the VDRis a key regulator ofthe canonical Wnt signalingpathway in keratinocyte stem cells and that these ligand-independent actions ofthe VDRare required for cutaneous homeostasis. The goal of these studies is not to study keratinocytebiologyper se, but rather to use this model system to characterize the molecularpathway and molecular partners involved in mediating these unique actions ofthe unliganded VDR. Thus, using the keratinocyte as a model,these studies will identify the novel molecular actions of the VDRwhich prevent the development of alopecia, a direct consequence ofVDR ablation.